The goal of the proposed project is to characterize pyruvate carboxylase (PC) deficiency at the molecular level. PC, a biotinylated mitochondrial enzyme, catalyzes the conversion to pyruvate to oxaloacetate. PC is a critical enzyme in intermediary metabolism as oxaloacetate is a substrate for both synthetic reactions such as gluconeogenesis and lipogenesis and anaplerotic reactions that replenish tricarboxylic acid and urea cycle intermediates. Defects in this enzyme effect the synthesis of neurotransmitters and myelin in the developing nervous system. Clinical manifestations of PC deficiency are heterogeneous and include mental retardation, developmental delay, neuroanatomic lesions, and lactic acidosis. The severity of this disorder varies with some children not surviving beyond the neonatal period while others have a more benign course. It has been hypothesized that severe PC occurs when the anaplerotic functions of this enzyme are affected. Molecular characterization of this disorder has been limited because the gene for human PC had not been characterized. Recently, a full-length human PC cDNA clone has been sequenced and the 5' regulatory region of the PC gene characterized. With this information, it is now possible to characterize PC deficiency at the molecular level. The specific aims of the proposed project are to 1) identify mutations responsible for PC deficiency and relate these alterations to clinical and biochemical phenotypes; 2) determine how natural and artificially induced mutations in critical regions of PC affect the structure and function of this enzyme by alterations in expression, assembly, and/or catalysis; and 3) assess the physiologic effects of PC deficiency on the anaplerotic functions of PC. The methods to be utilized in the proposed project include DNA sequencing, conformational analysis, in situ mutagenesis, protein expression in prokaryotic vectors, biochemical kinetic analysis, and stable isotope labeling of tricarboxylic acid cycle intermediates. The data collected from these studies will aid in determining the cause(s) for the clinical heterogeneity of this metabolic birth defect. The results of these studies will enhance our understanding of the etiology and pathophysiology of PC deficiency and facilitate improved diagnosis and treatment of this disorder.